PROJECT SUMMARY/ABSTRACT Arsenic and benzo[a]pyrene (BaP) are among the most common environmental pollutants and human exposure to arsenic and BaP mixture is very common. Our knowledge on the effect and mechanism of arsenic and BaP co- exposure is limited. The goal of this study is to determine the mechanism of the effect of arsenic and BaP co- exposure. Epigenetics refers to heritable changes in the pattern of gene expression that are not caused by changes in DNA sequence, but are mediated by DNA methylation, histone posttranslational modifications and noncoding RNAs. Cancer stem cells (CSCs) are cancer cells possessing characteristics of normal stem cells. CSCs or CSC- like cells are considered as cancer initiating cells. While the mechanism of arsenic toxic effect is not clearly defined, it is thought that the toxic effect of BaP mainly depends on its metabolic activation and DNA adduct formation. The reported effects of arsenic on DNA adduct level are controversial, suggesting that other mechanisms may play a key role in the combined effect of arsenic and BaP co-exposure. Our preliminary studies found: (i) Arsenic and BaP co-exposure has a synergistic effect in inducing cell transformation, CSC-like property and lung tumors; (ii) Arsenic and BaP co-exposure has no synergistic effect on BPDE-DNA adduct level. In contrast, the co-exposure shows a synergistic effect in epigenetic deregulations as evidenced by increasing the levels of histone H3K9 methyltransferase SUV39H1 and H3 repressive methylation mark H3K9me2. Moreover, the co-exposure also shows a synergistic effect in reducing the level of suppressor of cytokine signaling 3 (SOCS3) and increasing Akt and Erk1/2 activation; (iii) ShRNA knockdown SUV39H1 reduces H3K9me2 level, increases SOCS3 level, reduces Akt and Erk1/2 activation and soft agar colony formation and CSC-like property; (iv) Stably re-expressing SOCS3 reduces Akt, Erk1/2 activation, soft agar colony formation and CSC-like property; (v) Inhibiting Akt and Erk/1/2 reduces soft agar colony formation and CSC-like property; and (vi) Arsenic and BaP co-exposure significantly increases aryl hydrocarbon receptor (AhR) nuclear localization and siRNA knockdown AhR greatly reduces the level of SUV39H1 in arsenic and BaP co-exposure-transformed cells. Based on our preliminary data, our central hypothesis is: (i) Arsenic and BaP co-exposure up-regulates HMTase SUV39H1 via synergistically activating AhR; (ii) Up-regulation of SUV39H1 leads to SOCS3 down-regulation; and (iii) Down-regulation of SOCS3 causes a stronger Akt and Erk1/2 activation, which enhances cell transformation and CSC-like property thus increasing lung tumorigenesis. Three aims are proposed: Aim 1: Arsenic and BaP co-exposure significantly enhances cell transformation and tumorigenesis by up-regulating HMTase SUV39H1 via synergistically activating AhR. Aim 2: SUV39H1 up-regulation by arsenic and BaP co-exposure enhances cell transformation and tumorigenesis by down-regulating SOCS3 leading to stronger activation of Akt and Erk1/2. Aim 3: Simultaneously inactivating both Akt and Erk1/2 by a natural compound Withaferin A impairs the synergistic effect of arsenic and BaP co- exposure in inducing cell transformation and lung tumorigenesis in mice.